Polymorphism of myosin among skeletal muscle fiber types

An immunocytochemical approach was used to localize myosin with respect to individual fibers in rat skeletal muscle. Transverse cryostat sections of rat diaphragm, a fast-twitch muscle, were exposed to fluorescein-labeled immunoglobulin against purified chicken pectoralis myosin. Fluorescence microscopy revealed a differential response among fiber types, identified on the basis of mitochondrial content. All white and intermediate fiber but only about half of the red fiber reacted with his antimyosin. In addition, an alkali-stable ATPase had the same pattern of distribution among fibers, which is consistent with the existence of two categories of red fibers. The positive response of certain red fibers indicates either that their myosin has antigenic determinants in common with "white" myosin, or that the immunogen contained a "red" myosin. Myosin, extracted from a small region of the pectorlis which consists entirely of white fibers, was used to prepare an immunoadsorbent column to isolate antibodies specific for white myosin. This purified anti-white myosin reacted with the same fibers of the rat diaphragm that had reacted with the white, intermediate, and some red fibers are sufficiently homologous to share antigenic determinants. In a slow-twitch muscle, the soleus, only a minority of the fiber reacted with antipectoralis myosin. The majority failed to respond; hence, they are not equivalent to intermediate fibers of the diaphragm; despite their intermediate mitochondrial content. Immunocytochemical analysis of two different musles of the rat has demonstrated that more than one isoenzyme of myosin can exist in a single muscle, and that individual fiber types can be recognized by immunological differences in their myosin. We conclude that, in the rat diaphragm, there are at least two immunochemically distinct types of myosin and four types of muscle fibers: white, intermediate, and two red. We suggest that these fibers correspond to the four types of motor units described by Burke et al. (Burke, R. E., D. N. Levine, P. Tsairis, and F. E. Zajac, III 1973. J. Physiol. (Lond) 234:723-748.)in the cat gastrocnemius.`     

Curare-induced transformation of myosin pattern in developing skeletal muscle fibers

The effects of neuromuscular block on the pattern of distribution of myosin isozymes in developing skeletal muscle fibers was examined by immunocytochemistry. The homogeneous population of fibers in the anterior latissimus dorsi (ALD) of the 18-day chick embryo was converted by curare to a mosaic of at least two categories of fibers. Normally all fibers in this slow muscle reacted with antibodies against slow myosin (anti-ALD). They also reacted with an antibody specific for the alkali 1 light chain (anti-delta 1) but not the alkali 2 light chain (anti-delta 2) of fast myosin. After treatment with curare, which inhibits neuronal cell death and increases the number of axonal endings, ALD muscle fibers continued to react with anti-delta 1, but many now reacted with anti-delta 2 as well. The same fibers failed to react with anti-ALD. From this it can be concluded that the myosin in this population was converted to a type not normally present. The changes, therefore, are not merely a result of the preferential loss of a slow type of fiber, nor are they a result of delayed maturation. In contrast, curare had no apparent effect on the fast posterior latissimus dorsi (PLD). As in the normal muscle at 18 days, all fibers reacted strongly with anti-delta 1 and to variable degrees with anti-delta 2, and very few fibers reacted with anti-ALD. Our observations suggest that the dual response to antibodies against fast and slow myosin during development is not a necessary consequence of multiple axon terminals. We present evidence that curare induces the expression of a different myosin in the embryonic ALD, and we suggest that the selective transformation of the fiber population may be a manifestation of a change in composition of the motoneuron pool.     

Heterogeneity of fast-oxidative muscle fibers of chicken demonstrated by anti-myosin monoclonal antibodies

Summary The fiber type composition of two fast muscles of the chicken, namely, adductor superficialis (AS) and pectoralis major (PM) was examined by the histochemical myosin ATPase staining and immunochemical techniques using monoclonal antibodies (McAbs). Two new McAbs produced against the myosin of the anterior latissimus dorsi (ALD) muscle of the chicken and named ALD-122 and ALD-83 were characterized to be specific for myosin heavy chain (MHC) and for myosin light chain-1 respectively. They were used in conjunction with previously reported McAbs specific for slow MHC (ALD-47), fast MHC (MF-14) and fast light chain-2 (MF-5). By the histochemical ATPase test most muscle fibers of AS and PM muscles reacted as IIA and IIB respectively. By immunofluorescent staining with the anti-MHC McAbs, ALD-122, and MF-14, the fibers of AS, muscle showed remarkable heterogeneity whereas PM muscle fibers reacted, uniformly. Differences in the myosin light chain composition of AS and PM muscles were also found by SDS-gel electrophoresis and immunoblot analysis with the anti-light chain McAb, ALD-83. The study clearly indicated that the histochemically homogenous (type IIA) AS muscle is composed of several subpopulations of fibers which differ in their myosin composition and that this heterogeneity of the muscle is not simply due to presence of variable amounts of slow myosin in its fibers.     

Ultrastructural identification of muscle fiber types by immunocytochemistry

In a fast-twitch muscle, three types of fibers (red, intermediate, and white) can be distinguished on the basis of mitochondrial content. Red fibers, identified by abundant mitochondria, can be further differentiated on the basis of a positive or negative response to antibodies specific for white ("fast") myosin. Because there is also a difference in Z-line width among fibers of the same muscle, the possibility existed that the two red fibers, which differ in type of myosin, might also differ in dimensions of the Z line. We therefore examined, with the electron microscope, fibers which had been exposed to antibody against white myosin. In those fibers which react with the antibody, an electron-opaque band is evident in the H-band region, thereby distinguishing reactive from unreactive fibers. The red fiber can now be subdivided on the basis of a positive or negative response to anti-white myosin visualized directly with the electron microscope. Both categories of red fibers ("fast" and "slow") have wide Z lines, and thus are distinguished from white and intermediate fibers, which react with the antibody but which have narrow Z lines. On the basis of combined immunocytochemical and ultrastructural characteristics, four types of fibers can be recognized in a single muscle. Moreover, it is demonstrated here that a wide Z line does not necessarily imply a slow speed of contraction.     

Distribution and properties of myosin isozymes in developing avian and mammalian skeletal muscle fibers

Isozymes of myosin have been localized with respect to individual fibers in differentiating skeletal muscles of the rat and chicken using immunocytochemistry. The myosin light chain pattern has been analyzed in the same muscles by two-dimensional PAGE. In the muscles of both species, the response to antibodies against fast and slow adult myosin is consistent with the speed of contraction of the muscle. During early development, when speed of contraction is slow in future fast and slow muscles, all the fibers react strongly with anti-slow as well as with anti-fast myosin. As adult contractile properties are acquired, the fibers react with antibodies specific for either fast or slow myosin, but few fibers react with both antibodies. The myosin light chain pattern slow shows a change with development: the initial light chains (LC) are principally of the fast type, LC1(f), and LC2(f), independent of whether the embryonic muscle is destined to become a fast or a slow muscle in the adult. The LC3(f), light chain does not appear in significant amounts until after birth, in agreement with earlier reports. The predominance of fast light chains during early stages of development is especially evident in the rat soleus and chicken ALD, both slow muscles, in which LC1(f), is gradually replaced by the slow light chain, LC1(s), as development proceeds. Other features of the light chain pattern include an "embryonic" light chain in fetal and neonatal muscles of the rat, as originally demonstrated by R.G. Whalen, G.S. Butler- Browne, and F. Gros. (1978. J. Mol. Biol. 126:415-431.); and the presence of approximately 10 percent slow light chains in embryonic pectoralis, a fast white muscle in the adult chicken. The response of differentiating muscle fibers to anti-slow myosin antibody cannot, however, be ascribed solely to the presence of slow light chains, since antibody specific for the slow heavy chain continues to react with all the fibers. We conclude that during early development, the myosin consists of a population of molecules in which the heavy chain can be associated with a fast, slow, or embryonic light chain. Biochemical analysis has shown that this embryonic heavy chain (or chains) is distinct from adult fast or slow myosin (R.G. Whalen, K. Schwartz, P. Bouveret, S.M. Sell, and F. Gros. 1979. Proc. Natl. Acad. Sci. U.S.A. 76:5197-5201. J.I. Rushbrook, and A. Stracher. 1979. Proc Natl. Acad. Sci. U.S.A. 76:4331-4334. P.A. Benfield, S. Lowey, and D.D. LeBlanc. 1981. Biophys. J. 33(2, Pt. 2):243a[Abstr.]). Embryonic myosin, therefore, constitutes a unique class of molecules, whose synthesis ceases before the muscle differentiates into an adult pattern of fiber types.     

Myosin heavy chain expression during development and following denervation of fast fibers in the red strip of the chicken pectoralis

The myosin heavy chain composition of muscle fibers that comprise the red strip of the pectoralis major was determined at different stages of development and following adult denervation. Using a library of characterized monoclonal antibodies we found that slow fibers of the red strip do not react with antibodies to any of the fast myosin heavy chains of the superficial pectoralis. Immunocytochemical analysis of the fast fibers of the adult red strip revealed that they contain the embryonic fast myosin heavy chain rather than the adult pectoral isoform found throughout the adult white pectoralis. This was confirmed using immunoblot analysis of myosin heavy chain peptide maps. We show that during development of the red strip both neonatal and adult myosin heavy chains appear transiently, but then disappear during maturation. Furthermore, while the fibers of the superficial pectoralis reexpress the neonatal isoform as a result of denervation, the fibers of the red strip reexpress the adult isoform. Our data demonstrate a new developmental program of fast myosin heavy chain expression in the chicken and suggest that the heterogeneity of myosin heavy chain expression in adult fast fibers results from repression of specific isoforms by innervation.     

Differentiation of the anterior latissimus dorsi muscle of the chicken examined by anti-myosin monoclonal antibodies

Two new monoclonal antibodies (McAbs), ALD-180 and ALD-88, produced against the myosin of the slow anterior latissimus dorsi (ALD) muscle of the chicken are described. Their specificity for myosin heavy chain (MHC) was established by radioimmunoassay, immunoautoradiography, and immunofluorescence. They were used in conjunction with McAbs MF-14 and MF-30 (which have been characterized previously to be directed against MHC of the fast skeletal muscle) to examine the developmental changes of the chicken ALD muscle. At the 16-day embryonic, early posthatch, and adult stages the ALD muscle fibers differed in their reaction pattern with the McAbs; at the embryonic stage all fibers reacted strongly with ALD-180 and weakly with ALD-88 and MF-30; at the early posthatch stage there was a checkerboard pattern with many fibers not reacting with any of these three McAbs; and at the adult stage all fibers reacted strongly with ALD-180 and ALD-88 and weakly with MF-30. The MF-14 antibody did not react with ALD muscle at any developmental stage. The mature pattern of immunoreactivity of the ALD muscle fibers with the antibodies was established only after 9 weeks posthatch, and during this 9-week period the immunofluorescence changes were nonsynchronous. Based on immunocytochemical evidence of changes in myosin isoform expression, this study clearly demonstrates a distinctive neonatal (early posthatch) stage in the development of the chicken slow muscle.     

Immunochemical analysis of C-protein isoform transitions during the development of chicken skeletal muscle

Isoforms of C-protein in adult chickens which differ in fast (pectoralis major, PM) and slow (anterior latissimus dorsi, ALD) skeletal muscles can be distinguished immunochemically with monoclonal antibodies (McAbs) specific for the respective fast (MF-1) and slow (ALD-66) protein variants (Reinach et al., 1982 and 1983). The expression of these C-proteins during chick muscle development in vivo has been analyzed by immunoblot and immunofluorescence procedures. Neither MF-1 nor ALD-66 reacted with whole-cell lysates or myofibrils from PM of 12-day-old embryos. However, both McAbs bound to peptides of 145 kDa in PM from late embryonic and young posthatched chickens. All of the myofibers in these muscles reacted with both antibodies, but the binding of the anti-slow McAb (ALD-66) diminished progressively with age and was completely negative with PM by 2 weeks after hatching. In contrast, the ALD muscle from 17 days in ovo thru adulthood only reacted with ALD-66; no binding of MF-1 could be detected at these stages. Since both fast and slow myosin light chains (LC) coexist within embryonic pectoralis and ALD muscles (e.g., G. F. Gauthier, S. Lowey, P. A. Benfield, and A. W. Hobbs, 1982, J. Cell Biol.92, 471–484) yet segregate to specific fast and slow muscle fibers at different stages of development, the temporal transitions of C-protein and myosin LC were compared during myogenesis. “Slow-type” C-protein appeared after the disappearance of slow myosin light chains, whereas the accumulation of the “fast-type” light chains occurred before the expression of “fast-type” C-protein. The pattern of isoform transitions appears to be far more complex than previously suspected.     

Characterization of the C-protein from posterior latissimus dorsi muscle of the adult chicken: heterogeneity within a single sarcomere

Specific isoforms of myofibrillar proteins are expressed in different muscles and in various fiber types within a single muscle. We have isolated and characterized monoclonal antibodies against C-proteins from slow tonic (anterior latissimus dorsi, ALD) and fast twitch (pectoralis major) muscles of the chicken. Although the antibody against "fast" C-protein (MF-1) did not bind to the "slow" isoform and the antibody to the "slow" C-protein (ALD-66) did not bind to the "fast" isoform, we observed that both antibodies bound C-protein from the posterior latissimus dorsi (PLD) muscle. Here we demonstrate that in the PLD muscle the binding sites of these two antibodies reside in two different C-protein isoforms which have different molecular weights and can be separated by hydroxylapatite column chromatography. Since we have shown previously that both these antibodies stain all myofibers and myofibrils derived from PLD muscle, we conclude that all myofibers in this muscle contain both isoforms with all sarcomeres.     

Myosin isozymes in normal and cross-reinnervated cat skeletal muscle fibers

Immunocytochemical characteristics of myosin have been demonstrated directly in normal and cross-reinnervated skeletal muscle fibers whose physiological properties have been defined. Fibers belonging to individual motor units were identified by the glycogen-depletion method, which permits correlation of cytochemical and physiological data on the same fibers. The normal flexor digitorum longus (FDL) of the cat is composed primarily of fast-twitch motor units having muscle fibers with high myosin ATPase activity. These fibers reacted with antibodies specific for the two light chains characteristic of fast myosin, but not with antibodies against slow myosin. Two categories of fast fibers, corresponding to two physiological motor unit types (FF and FR), differed in their immunochemical response, from which it can be concluded that their myosins are distinctive. The soleus (SOL) consists almost entirely of slow-twitch motor units having muscle fibers with low myosin ATPase activity. These fibers reacted with antibodies against slow myosin, but not with antibodies specific for fast myosin. When the FDL muscle was cross-reinnervated by the SOL nerve, twitch contraction times were slowed about twofold, and motor units resembled SOL units in a number of physiological properties. The corresponding muscle fibers had low ATPase activity, and they reacted with antibodies against slow myosin only. The myosin of individual cross-reinnervated FDL muscle units was therefore transformed, apparently completely, to a slow type. In contrast, cross-reinnervation of the SOL muscle by FDL motoneurons did not effect a complete converse transformation. Although cross-reinnervated SOL motor units had faster than normal twitch contraction times (about twofold), other physiological properties characteristic of type S motor units were unchanged. Despite the change in contraction times, cross-reinnervated SOL muscle fibers exhibited no change in ATPase activity. They also continued to react with antibodies against slow myosin, but in contrast to the normal SOL, they now showed a positive response to an antibody specific for one of the light chains of fast myosin. The myosins of both fast and slow muscles were thus converted by cross-reinnervation, but in the SOL, the newly synthesized myosin was not equivalent to that normally present in either the FDL or SOL. This suggests that, in the SOL, alteration of the nerve supply and the associated dynamic activity pattern are not sufficient to completely respecify the type of myosin expressed.     

Muscle Fiber Types in the Pectoralis of the White Pelican,a Soaring Bird

The pectoralis muscle (M. pectoralis) of many premier soaring birds contains a smaller, accessory, deep belly in addition to the much larger superficial belly found in all flying birds. Here we describe the muscle fiber types in both the superficial and deep bellies of the pectoralis of one such adept soaring species, the white pelican (Pelecanus erythrorhynchos).Histochemical techniques are used to demonstrate both nicotinamide adenine dinucleotide (reduced) and myofibrillar adenosine triphosphatase activities within the muscle fibers. Immunocytochemical methods employing several monoclonal antibodies, each directed against a different myosin heavy chain epitope of the chicken, are also used to characterize the fibers. While the superficial belly of the muscle consists entirely of fast-twitch oxidative-glycolytic fibers, the deep belly is composed exclusively of slow fibers. These slow fibers are labelled by two different antibodies specific for chicken slow myosin. We suggest that the fibers of the superficial belly are best suited to flapping flight, and that the fibers of the deep belly would be recruited only during soaring flight. Furthermore, we hypothesize that the deep belly found in the pectoralis of soaring species probably evolved from a deep neuromuscular compartment of the superficial belly.     

Skeletal muscle satellite cell diversity: satellite cells form fibers of different types in cell culture

Following skeletal muscle injury, new fibers form from resident satellite cells which reestablish the fiber composition of the original muscle. We have used a cell culture system to analyze satellite cells isolated from adult chicken and quail pectoralis major (PM; a fast muscle) and anterior latissimus dorsi (ALD; a slow muscle) to determine if satellite cells isolated from fast or slow muscles produce one or several types of fibers when they form new fibers in vitro in the absence of innervation or a specific extracellular milieu. The types of fibers formed in satellite cell cultures were determined using immunoblotting and immunocytochemistry with monoclonal antibodies specific for avian fast and slow myosin heavy chain (MHC) isoforms. We found that satellite cells were of different types and that fast and slow muscles differed in the percentage of each type they contained. Primary satellite cells isolated from the PM formed only fast fibers, while up to 25% of those isolated from ALD formed fibers that were both fast and slow (fast/slow fibers), the remainder being fast only. Fast/slow fibers formed from chicken satellite cells expressed slow MHC1, while slow MHC2 predominated in fast/slow fibers formed from quail satellite cells. Prolonged primary culture did not alter the relative proportions of fast to fast/slow fibers in high density cultures of either chicken or quail satellite cells. No change in commitment was observed in fibers formed from chicken satellite cell progeny repeatedly subcultured at high density, while fibers formed from subcultured quail satellite cell progeny demonstrated increasing commitment to fast/slow fiber type formation. Quail satellite cells cloned from high density cultures formed colonies that demonstrated a similar change in commitment from fast to fast/slow, as did serially subcloned individual satellite cell progeny, indicating that the observed change from fast to fast/slow differentiation resulted from intrinsic changes within a satellite cell. Thus satellite cells freshly isolated from adult chicken and quail are committed to form fibers of at least two types, satellite cells of these two types are found in different proportions in fast and slow muscles, and repeated cell proliferation of quail satellite cell progeny may alter satellite cell progeny to increasingly form fibers of a single type.     

Immunocytochemical localization of aldolase in normal, denervated, and dystrophic chicken muscles

To investigate whether immunocytochemical localization of muscle-specific aldolase can be used for fiber phenotype determination, we produced specific antibodies against the enzyme and studied its distribution in adult chicken skeletal muscles by indirect immunofluorescence microscopy. Monoclonal antibodies against the myosin heavy chains of fast-twitch (MF-14) and slow-tonic (ALD-58) muscle fibers were also used to correlate aldolase levels with the fiber phenotype. The goat anti-aldolase antibody was found to be specific for the A form of aldolase, as evidenced by sodium dodecyl sulfate gel electrophoresis, immunotitration experiments, and immunoblot analysis. The antibody reacted strongly with the fast-twitch myofibers of normal pectoralis and posterior latissimus dorsi muscles; the phenotype of these muscle fibers was confirmed by a positive immunofluorescent reaction after incubation with MF-14 antibody. By contrast, the slow-tonic myofibers of normal anterior latissimus dorsi, which react positively with ALD-58 antibody, reacted weakly with anti-aldolase antibodies. In denervated chicken muscles, reaction to anti-aldolase antibodies was markedly reduced in fast-twitch fibers, although reaction to MF-14 was not diminished. By contrast, in dystrophic muscle, fast-twitch fibers showed reduced reactivity to anti-aldolase and marked to moderate reduction in MF-14 reactivity. Our results show that: (a) in normal muscles, reactivity to anti-aldolase matches the phenotype obtained by using anti-fast or anti-slow myosin heavy chain antibodies, and therefore can serve to identify mature fibers as fast or slow; and (b) in denervated or dystrophic muscles, the intracellular expressions of aldolase and fast-twitch myosin heavy chains are regulated independently.     

Fiber-type proportions in mammalian soleus muscle during postnatal development

We analyzed the fiber-type composition of the soleus muscle in rats and mice to determine whether the adult proportion of fiber types is fixed soon after birth or whether it changes during postnatal maturation. We examined muscles from animals varying in age from 1 week to 1 year using monoclonal antibodies that distinguish between fast and slow isoforms of myosin heavy chains. In cross sections of unfixed muscle containing profiles of all myofibers in the muscle, we counted the fibers that stained with antibodies to fast myosin, and in adjacent sections, those that stained positive with an antibody to slow myosin. We also counted the total number of fibers in each section. Rat soleus contained about 2500 myofibers, and mouse about 1000 at all ages studied, suggesting that myogenesis ceases in soleus by 1 week after birth or sooner. In mouse soleus, the relative proportions of fibers staining positive with fast and slow myosin antibodies were similar at all ages studied, about 60%–70% being fast and 30%–40% slow. In rat soleus, however, the proportions of fast antibody-positive and slow antibody-positive fibers changed dramatically during postnatal maturation. At 1 week after birth, about 50% of rat soleus fibers stained with fast myosin antibodies, whereas between 1 and 2 months this value fell to about 10%. In mouse, about 10% of fibers at 1 week, but none at 1 year, reacted with both fast and slow antibodies, whereas in rat, fewer than 3% bound both antibodies to a significant degree at 1 week. It is puzzling why, in rat soleus, the majority of apparently fast fibers present at 1 week is converted to a slow phenotype, whereas in mouse soleus the predominant change appears to be the suppression of fast myosin expression in a subset of fibers that expresses both myosin types at 1 week. It is possible that this may be related to differences in size and the amount of body growth between these two species.     

Fiber-type proportions in mammalian soleus muscle during postnatal development.

We analyzed the fiber-type composition of the soleus muscle in rats and mice to determine whether the adult proportion of fiber types is fixed soon after birth or whether it changes during postnatal maturation. We examined muscles from animals varying in age from 1 week to 1 year using monoclonal antibodies that distinguish between fast and slow isoforms of myosin heavy chains. In cross sections of unfixed muscle containing profiles of all myofibers in the muscle, we counted the fibers that stained with antibodies to fast myosin, and in adjacent sections, those that stained positive with an antibody to slow myosin. We also counted the total number of fibers in each section. Rat soleus contained about 2500 myofibers, and mouse about 1000 at all ages studied, suggesting that myogenesis ceases in soleus by 1 week after birth or sooner. In mouse soleus, the relative proportions of fibers staining positive with fast and slow myosin antibodies were similar at all ages studied, about 60%-70% being fast and 30%-40% slow. In rat soleus, however, the proportions of fast antibody-positive and slow antibody-positive fibers changed dramatically during postnatal maturation. At 1 week after birth, about 50% of rat soleus fibers stained with fast myosin antibodies, whereas between 1 and 2 months this value fell to about 10%. In mouse, about 10% of fibers at 1 week, but none at 1 year, reacted with both fast and slow antibodies, whereas in rat, fewer than 3% bound both antibodies to a significant degree at 1 week. It is puzzling why, in rat soleus, the majority of apparently fast fibers present at 1 week is converted to a slow phenotype, whereas in mouse soleus the predominant change appears to be the suppression of fast myosin expression in a subset of fibers that expresses both myosin types at 1 week. It is possible that this may be related to differences in size and the amount of body growth between these two species.     

A combined histochemical and immunohistochemical study on the dynamics of fast-to-slow fiber transformation in chronically stimulated rabbit muscle

Summary Chronically stimulated fast-twitch muscles of the rabbit were histochemically and immunohistochemically analyzed in serial cross sections (1) for percentages of fiber types, and (2) for the presence of myosin heavy chain isoforms during fast-to-slow transformation. By four weeks of stimulation the number of type-I fibers had increased more than fourfold, while only about 6% of the original IIB fibers remained. Type-IC and -IIC fibers transiently rose to 20% of the total fiber population. After 16 weeks, the number of type-I fibers had increased to 42%. With prolonged stimulation fewer fibers reacted with antibodies against embryonic and neonatal myosins and more with the antibody against slow myosin. The reaction for embryonic myosin was most often detected in the C fibers (IC, IIC). Immunohistochemical subtypes were observed for each fiber type in the stimulated muscles. The greatest number was seen in type-IIC fibers, which, in addition to their reaction for fast/neonatal and slow myosins, might also react with the antibodies against neonatal/embryonic and embryonic myosins. These findings indicated that the transforming fibers temporarily expressed myosin heavy chain isoforms normally not detectable in adult skeletal muscle. Myotubes reacted strongly with the antibodies against fast/neonatal and embryonic myosins, and some of them also with the antibody against slow myosin. Thus, it appears that under the influence of the low frequency stimulus pattern some of the newly formed myotubes developed into type-I fibers.     

Diversity in expression of myosin heavy chain isoforms and M-band proteins in rat muscle spindles

The composition of adult rat soleus muscle spindles, with respect to myosin heavy chain isoforms and M-band proteins, was studied by light-microscope immunohistochemistry. Serial sections were labelled with antibodies against slow tonic, slow twitch, fast twitch and neonatal myosin isoforms as well as against myomesin, M-protein and the MM form of creatine kinase. Intrafusal fiber types were distinguished according to the pattern of ATPase activity following acid and alkaline preincubations. Nuclear bag1 fibers were always strongly stained throughout with anti-slow tonic myosin, were positive for anti-slow twitch myosin towards and in the C-region but were unstained with anti-fast twitch and anti-neonatal myosins. The staining of nuclear bag2 fibers was in general highly variable. However, they were most often strongly stained by anti-slow tonic myosin in the A-region and gradually lost this reactivity towards the poles, whereas a positive reaction with anti-slow twitch myosins was found along the whole fiber. Regional staining variability with anti-neonatal and anti-fast myosins was apparent, often with decreasing intensity towards the polar regions. Nuclear chain fibers showed strong transient reactivity with anti-slow tonic myosin in the equatorial region, did not react with anti-slow twitch and were always evenly stained by anti-fast twitch and anti-neonatal myosins. All three intrafusal fiber types were stained with anti-myomesin. Nuclear bag1 fibers lacked staining for M-protein, whereas bag2 fibers displayed intermediate staining, with regional variability, often increasing in reactivity towards the polar regions. Chain fibers were always strongly stained by anti-M-protein. The MM form of creatine kinase was present in all three fiber types, but bag1 fibers were less reactive and clear striations were not observed, in contrast to bag2 and chain fibers. Out of 38 cross sectioned spindles two were found to have an atypical fiber composition (lack of chain fibers) and a rather diverse staining pattern for the different antibodies tested. Taken together, the data show that in adult rat soleus, slow tonic and neonatal myosin heavy chain isoforms are only expressed in the muscle spindle fibers and that each intrafusal fiber type has a unique, although variable, composition of myosin heavy chain isoforms and M-band proteins. We propose that both motor and sensory innervation might be the determining factors regulating the variable expression of myosin heavy chain isoforms and M-band proteins in intrafusal fibers of rat muscle spindles.     

Diversity in expression of myosin heavy chain isoforms and M-band proteins in rat muscle spindles

Summary The composition of adult rat soleus muscle spindles, with respect to myosin heavy chain isoforms and M-band proteins, was studied by light-microscope immunohistochemistry. Serial sections were labelled with antibodies against slow tonic, slow twitch, fast twitch and neonatal myosin isoforms as well as against myomesin, M-protein and the MM form of creatine kinase. Intrafusal fiber types were distinguished according to the pattern of ATPase activity following acid and alkaline preincubations.Nuclear bag₁ fibers were always strongly stained throughout with anti-slow tonic myosin, were positive for anti-slow twitch myosin towards and in the C-region but were unstained with anti-fast twitch and anti-neonatal myosins. The staining of nuclear bag₂ fibers was in general highly variable. However, they were most often strongly stained by anti-slow tonic myosin in the A-region and gradually lost this reactivity towards the poles, whereas a positive reaction with anti-slow twitch myosins was found along the whole fiber. Regional staining variability with antineonatal and anti-fast myosins was apparent, often with decreasing intensity towards the polar regions. Nuclear chain fibers showed strong transient reactivity with anti-slow tonic myosin in the equatorial region, did not react with anti-slow twitch and were always evenly stained by anti-fast twitch and anti-neonatal myosins. All three intrafusal fiber types were stained with anti-myomesin. Nuclear bag₁ fibers lacked staining for M-protein, whereas bag₂ fibers displayed intermediate staining, with regional variability, often increasing in reactivity towards the polar regions. Chain fibers were always strongly stained by anti-M-protein. The MM form of creatine kinase was present in all three fiber types, but bag₁ fibers were less reactive and clear striations were not observed, in contrast to bag₂ and chain fibers. Out of 38 cross sectioned spindles two were found to have an atypical fiber composition, (lack of chain fibers) and a rather diverse staining pattern for the different antibodies tested.Taken together, the data show that in adult rat solcus, slow tonic and neonatal myosin heavy, chain isoforms are only expressed in the muscle spindle fibers and that each intrafusal fiber type has a unique, although variable, composition of myosin heavy chain isoforms and M-band proteins. We propose that both motor and sensory innervation might be the determining factors regulating the variable expression of myosin heavy chain isoforms and M-band proteins in intrafusal fibers of rat muscle spindles.     

Regional differences in the expression of myosin light chains and tropomyosin subunits during development of chicken breast muscle

Types of myosin light chains and tropomyosins present in various regions and at different developmental stages of embryonic and posthatched chicken breast muscle (pectoralis major) have been characterized by two-dimensional gel electrophoresis. In the embryonic muscle all areas appear to accumulate both slow and fast forms of mysoin light chains in addition to α and β forms of tropomyosin. During development regional differences in myosin and tropomyosin expression become apparent. Slow myosin subunits become gradually restricted to areas of the anterior region of the muscle and finally become localized to a small red strip found on its anterior deep surface. This red region is characterized by the presence of slow and fast myosin light chains, α-fast, α-slow, and β-tropomyosin. In all other areas of the muscle examined only fast myosin light chains, β-tropomyosin and the α-fast form of tropomyosin, are found. In addition, β-tropomyosin also gradually becomes lost in the posterior regions of the developing breast muscle. In the adult, the red strip area represents less than 1% of the total pectoralis major mass and of the myosin extracted from this area approximately 15% was present as an isozyme that comigrated on nondenaturing gels with myosin from a slow muscle (anterior latissimus dorsi). The red region accumulates therefore fast as well as slow muscle myosin. Thus while the adult chicken pectoralis major is over 99% fast white muscle, the embryonic muscle displays a significant and changing capacity to accumulate both fast and slow muscle peptides.     

Myosin types in cultured muscle cells

Fluorescent antibodies against fast skeletal, slow skeletal, and ventricular myosins were applied to muscle cultures from embryonic pectoralis and ventricular myocadium of the chicken. A number of spindle-shaped mononucleated cells, presumably myoblasts, and all myotubes present in skeletal muscle cultures were labeled by all three antimyosin antisera. In contrast, in cultures from ventricular myocardium all muscle cells were labeled by anti-ventricular myosin, whereas only part of them were stained by anti-slow skeletal myosin and rare cells reacted with anti-fast skeletal myosin. The findings indicate that myosin(s) present in cultured embryonic skeletal muscle cells contains antigenic determinants similar to those present in adult fast skeletal, slow skeletal, and ventricular myosins.